Bioreactor array and methods of combinatorial testing

ABSTRACT

Methods and apparatus for culturing microorganisms are described, including culturing in mixotrophic culture conditions. A bioreactor array with multiple culture vessels with independently controllable inputs is used to culture similar cultures of microorganisms in which at least one parameter differs from other culture vessels in the bioreactor array.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. patent application Ser. No.14/805,267, filed Jul. 21, 2015, entitled Bioreactor Array and Methodsof Combinatorial Testing, and PCT Application No. PCT/US2014/016462,filed Feb. 14, 2014, entitled Bioreactor Array and Methods ofCombinatorial Testing, U.S. Provisional Application No. 61/850,623,filed Feb. 19, 2013, entitled Photobioreactor Array and MixotrophicCulture, and U.S. Provisional Application No. 61/917,423, filed Dec. 18,2013, entitled Photobioreactor Array and Mixotrophic Culture, the entirecontents of which are hereby incorporated by reference.

BACKGROUND

Photosynthetic organisms have been cultured to produce chemicals andbiologicals of interests such as fatty acids, proteins, hydrogen gas,pigments, carbohydrates, sugars, and vitamins for use in food, feed,pharmaceuticals, nutraceuticals, fuels, and other products.

Of particular interest in recent years has been the use ofmicroorganisms, such as microalgae and photosynthetic bacteria cultures,themselves or extracts derived from the microorganism. A number ofmicroalgal metabolites have commercial interest as chemical compoundsand have been so produced. Many have attempted to grow microalgae,typically in open ponds, long tubes, and bags with tubes between them.Others have attempted growing microalgae in enclosed tank systems, buteach type of system has encountered difficulties related to control andoptimization of the microalgae culture. Heterotrophic microorganisms,such as microalgae and bacteria, have also been cultured in traditionalstainless steel fermenters to produce for similar chemicals andbiologicals for commercial products in the same fields.

In an attempt to enhance production of a microalgae culture, a parameterof the biocultivation process has been varied in an attempt to find theoptimum range for that parameter.

However, it is known that multiple parameters interact with each otherwithin a culture to give complex relationships regarding optimal rangesfor each parameter. Even a simple component, such as the culture mediumunder auxotrophic conditions containing no more than salts, lackssimplistic optimization. For example, Pandey et al, Journal of AlgalBiomass Utilization, 1(3) p. 70-81 (2010), reports very different yieldsusing differing proportions of salts in their culturing medium even withall other culture parameters remaining unchanged. Within the article, itis noteworthy that no single salt concentration had an optimal rangethat controlled yield. Even different conventional culture mediaresulted in doubling the yield. The media compared were all previouslypublished for Spirulina and previously optimized for Spirulina growth,yet when compared side-by-side, dramatically different yields resulted.

Given the potential market, the need for a large-scale microalgae growthsystem is evident; yet, the prior art systems have had littleoptimization before being built. Different bioreactors present differentproblems and are optimized differently around the parameters which arenot changeable. Open ponds suffer from problems with contamination(e.g., organics, bacteria, fungi), changing conditions throughout theday and night (e.g., temperature, sunlight, wind), and other suboptimalcultivation conditions. Many bioreactors have difficulties withsufficient light reaching the microalgae for photosynthetic activity andin controlling the conditions (e.g., temperature, pH, nitrate levels). Asystem relying solely on ambient light as an energy source for themicroalgae is susceptible to fluctuation with seasons and even clouds,which may vary random and non-reproducible manner making comparisonbetween different runs difficult or impossible to interpret even whenthe same bioreactor is used.

Therefore, there is a need in the art for a system and method ofoptimizing culturing parameters to increase the efficiency of microalgaecultivation.

SUMMARY

The present invention provides a method and apparatus for cultivatingmicroorganisms in a liquid or aqueous culture medium with control overthe culturing parameters.

In one embodiment, a bioreactor array may comprise a plurality ofculture vessels configured to contain an aqueous culture ofmicroorganisms in an interior volume, each culturing vessel comprisingan independently controllable: gas supply, nutrient supply, heatexchanger, harvesting mechanism, and light supply; at least one sensorconfigured to measure at least one culturing parameter of each culturevessel; and a programmable logic controller. In some embodiments theculture vessel may have a volume of 500 to 1000 ml. In some embodiments,the culture vessel may be transparent. In some embodiments, the culturevessel may comprise at least one opaque section.

In some embodiments, the gas supply may comprise at least one gasselected from the group consisting of air, carbon dioxide, oxygen, andnitrogen. In some embodiments, the nutrient supply may comprise anorganic carbon supply. In some embodiments, the harvesting system maycomprise an overflow system configured to passively remove at least partof the aqueous culture volume at a culture volume less than a totalvolume of the culture vessel. In some embodiments, the at least onesensor may be selected from the group consisting of: pH sensor,temperature sensors, light sensors, dissolved oxygen sensors, dissolvedcarbon dioxide sensor, and optical density sensor. In some embodiments,the light supply may comprise a lighting device disposed outside of theculture vessel. In some embodiments, the light supply may comprise alighting device disposed in the interior volume of the culture vessel.

In another embodiment, a method of culturing microorganisms may compriseproviding culture of microorganisms in an aqueous culture medium in aplurality of culture vessels; independently controlling the supply toeach culture vessel at least one from the group consisting of: light, atleast one gas, at least one nutrient, and heat exchange; and whereineach culture vessel contains a culture of the same microorganismscultured with different parameters selected from the group consisting oftemperature, pH, amount of light, intensity of light, wavelengths oflight, light photoperiod, light/dark cycle, concentrations of gases, andagitation from gas supply. In some embodiments, the at least onenutrient may comprise organic carbon.

In some embodiments, the method may further comprise harvesting at leastpart of the aqueous culture from the culture volume. In someembodiments, the different parameters may further comprise harvestrates. In some embodiments, the culture vessel may be supplied withlight and organic carbon. In some embodiments, the culture may besupplied with organic carbon but no light.

In some embodiments, providing the culture of microorganisms in theaqueous culture medium in each culture vessel may comprise 0.5-1.5 gramsof biomass. In some embodiments, providing the culture of microorganismsin the aqueous culture medium in each culture vessel comprises 500 to1,000 ml of culture volume. In some embodiments, the method may furthercomprise monitoring the parameters of the culture of microorganisms inthe plurality of culture vessels with at least one sensors and aprogrammable logic controller. In some embodiments, the supply of lightmay comprise at least two independently controllable light emittingdiodes providing different wavelengths of light.

The current invention provides a method for strain conditioning,adaptation and selection. The method may be performed in a plurality ofbioreactors where at least one parameter stressing the microorganismculture differs between bioreactors. Alternatively, the inventivereactors may be used to evaluate growth parameters or treatments(chemical or biological) to control or reduce contamination inmicroorganism cultures.

In another embodiments, the bioreactor array may comprise: a pluralityof culture vessels configured to contain an aqueous culture ofmicroorganisms in an interior volume, each culturing vessel comprisingmeans for controlling the gas composition, nutrient composition,temperature, light exposure, and harvest of the aqueous culture ofmicroorganisms; and means for measuring at least one culturing parameterof each culture vessel. In some embodiments, the bioreactor array mayfurther comprise computer automated means for controlling at least oneof the gas composition, nutrient composition, temperature, lightexposure, and harvest of the aqueous culture of microorganisms inresponse to the at least one culturing parameters. In some embodimentsthe at least one culturing parameter may comprise at least one from thegroup consisting of: nutrient concentration, temperature, pH, amount oflight, intensity of light, wavelengths of light, light photoperiod,light/dark cycle, concentrations of gases, and agitation from gas supply

DETAILED DESCRIPTION

While not wishing to be bound by any particularly theory, the presentinvention is believed to determine desirable effects on microorganisms,including photosynthetic microorganisms, and/or products as the resultof the metabolism of microorganisms used.

For the purposes of this specification the term “photosyntheticmicroorganism” is intended to cover any phototrophic or mixotrophic ormicroorganism that is capable of utilizing light as a source of energythrough photosynthesis. The photosynthesis need not be directly involvedin producing the desired result. The photosynthesis need not even occurprovided that an alternative energy source is provided. All organismsthat utilize light for photosynthesis, of which phototrophic andmixotrophic species of microorganism are included.

The term “microorganism” refers to microscopic organisms such asmicroalgae and cyanobacteria. Microalgae include microscopicmulti-cellular plants (e.g. duckweed), photosynthetic microorganisms,heterotrophic microorganisms, diatoms, dinoflagelattes, and unicellularalgae.

The terms “microbiological culture”, “microbial culture”, or“microorganism culture” refer to a method or system for multiplyingmicroorganisms through reproduction in a predetermined culture medium,including under controlled laboratory conditions. Microbiologicalcultures, microbial cultures, and microorganism cultures are used tomultiply the organism, to determine the type of organism, or theabundance of the organism in the sample being tested. In liquid culturemedium, the term microbiological, microbial, or microorganism culturegenerally refers to the entire liquid medium and the microorganisms inthe liquid medium regardless of the vessel in which the culture resides.A liquid medium is often referred to as “media”, “culture medium”, or“culture media”. The act of culturing is generally referred to as“culturing microorganisms” when emphasis is on plural microorganisms.The act of culturing is generally referred to as “culturing amicroorganism” when importance is placed on a species or genus ofmicroorganism. Microorganism culture is used synonymously with cultureof microorganisms.

The terms “phototrophic”, “phototrophy”, “photoautotrophy”,“photoautotrophic”, and “autotroph” refer to culture conditions in whichlight and inorganic carbon (e.g., carbon dioxide, carbonate,bi-carbonate) may be applied to a culture of microorganisms.Microorganisms capable of growing in phototrophic conditions may uselight as an energy source and inorganic carbon (e.g., carbon dioxide) asa carbon source. A microorganism in phototrophic conditions may produceoxygen.

The terms “mixotrophic” and “mixotrophy” refer to culture conditions inwhich light, organic carbon, and inorganic carbon (e.g., carbon dioxide,carbonate, bi-carbonate) may be applied to a culture of microorganisms.Microorganisms capable of growing in mixotrophic conditions have themetabolic profile of both phototrophic and heterotrophic microorganisms,and may use both light and organic carbon as energy sources, as well asboth inorganic carbon and organic carbon as carbon sources. Amixotrophic microorganism may be using light, inorganic carbon, andorganic carbon through the phototrophic and heterotrophic metabolismssimultaneously or may switch between the utilization of each metabolism.A microorganism in mixotrophic culture conditions may be a net oxygen orcarbon dioxide producer depending on the energy source and carbon sourceutilized by the microorganism. Microorganisms capable of mixotrophicgrowth comprise microorganisms with the natural metabolism and abilityto grow in mixotrophic conditions, as well as microorganisms whichobtain the metabolism and ability through modification of cells by wayof methods such as mutagenesis or genetic engineering.

The terms “heterotrophic” and “heterotrophy” refer to culture conditionsin which organic carbon may be applied to a culture of microorganisms inthe absence of light. Microorganisms capable of growing in heterotrophicconditions may use organic carbon as both an energy source and as acarbon source. A microorganism in heterotrophic conditions may producecarbon dioxide.

The organic carbon sources suitable for growing a microorganismmixotrophically may comprise: acetate, acetic acid, ammonium linoleate,arabinose, arginine, aspartic acid, butyric acid, cellulose, citricacid, ethanol, fructose, fatty acids, galactose, glucose, glycerol,glycine, lactic acid, lactose, maleic acid, maltose, mannose, methanol,molasses, peptone, plant based hydrolyzate, proline, propionic acid,ribose, sacchrose, partial or complete hydrolysates of starch, sucrose,tartaric, TCA-cycle organic acids, thin stillage, urea, industrial wastesolutions, yeast extract, and combinations thereof. The organic carbonsource may comprise any single source, combination of sources, anddilutions of single sources or combinations of sources.

Of particular use in the present invention are mixotrophicmicroorganisms such as, but not limited to Agmenellum, Amphora,Anabaena, Anacystis, Apistonema, Pleurochyrsis, Arthrospira (Spirulina),Botryococcus, Brachiomonas, Chlamydomonas, Chlorella, Chloroccum,Cruciplacolithus, Cylindrotheca, Coenochloris, Cyanophora, Cyclotella,Dunaliella, Emiliania, Euglena, Extubocellulus, Fragilaria, Galdieria,Goniotrichium, Haematococcus, Halochlorella, Isochyrsis, Leptocylindrus,Micractinium, Melosira, Monodus, Nostoc, Nannochloris, Nannochloropsis,Navicula, Neospongiococcum, Nitzschia., Odontella, Ochromonas,Ochrosphaera, Pavlova, Picochlorum, Phaeodactylum, Pleurochyrsis,Porphyridium, Poteriochromonas, Prymnesium, Rhodomonas, Scenedesmus,Skeletonema, Spumella, Stauroneis, Stichococcus, Auxenochlorella,Cheatoceros, Neochloris, Ocromonas, Porphiridium, Synechococcus,Synechocystis, Tetraselmis, Thraustochytrids, Thalassiosira, and speciesthereof. Also included may be more unclassified new microalgal genera,species, or strains which may be poorly characterized or even newlydiscovered microorganisms. While certain high yielding strains arepreferentially used, a considerable number of other organisms are knownand under the property conditions may also produce biomass and variousmetabolites of interest. National Renewable Energy Laboratory (NREL) hasselected 300 species of microalgae, both fresh water and salt watermicroalgae, including diatoms and green microalgae. Other organizationshave large depositories of photosynthetic and non-photosyntheticmicroorganisms. Any of these and others may be used in the presentinvention. Known literature also describes a plurality of microorganismscapable of growth in phototrophic and heterotrophic culture conditionswhich is also of interest with this invention.

For the purposes of this specification the term “parameter” refers toany feature of the culturing of the microorganism or extraction orprocessing of the resulting culture. Examples may comprise: bioreactordesign; microorganism strain selection; additional microorganism strainselection (if mixed culture); selection of media components andconcentration of each; dissolved gases; gases in the atmosphere abovethe microorganism culture; carbon dioxide, oxygen and other gas amountsand pressures in the bioreactor; gas sparaging rate; gas bubble size;chemical carbon source (if any); content of, timing of and rate ofaddition of additional nutrients (e.g., organic carbon); fluid withdrawrate (i.e., harvesting rate); culture mixing rate; cell withdraw rate;initial cell concentration; maintenance of steady cell concentration;pH; salinity; osmotic pressure; temperature; amount of light;wavelength(s) of light; pulsing duration and rate of light; length oflight and dark cycles; rate of increasing and decreasing light intensityduring the lighting phase; selection of a chemical enhancing productproduction or its amount or timing of its addition; selection ofchemical or physical inhibition of contaminating microorganisms (amountand timing also); manner of and rate of removal of wastes orcontaminants; choice of added contaminant or proxy to mimic naturalcontamination; type of culture operation (e.g., batch, semi-batch,fed-batch, continuous); rate and timing for removal of secretedproduct(s); longevity of the culture; co-cultivation strategies andstrain interactions; and timing of a change in one or more parameter inresponse to a set schedule or in response to a change in condition ofone or more other condition.

The term “axenic” describes a culture of an organism that is entirelyfree of all other “contaminating” organisms (i.e., organisms that aredetrimental to the health of the microalgae or cyanobacteria culture).Throughout the specification, axenic refers to a culture that wheninoculated in an agar plate with bacterial basal medium, does not formany colonies other than the microorganism of interest. Axenic describescultures not contaminated by or associated with any other livingorganisms such as but not limited to bacteria, cyanobacteria, microalgaeand/or fungi. Axenic is usually used in reference to pure cultures ofmicroorganisms that are completely free of the presence of otherdifferent organisms. An axenic culture of microalgae or cyanobacteria iscompletely free from other different organisms.

Should one seek to optimize multiple parameters, the combinationsindicate one will generate a large set of test data of different cultureparameters. This is particularly challenging for mixotrophic cultureswith many additional complicating factors. This is impractical toattempt in a mass production system as it may take years of testing tooptimize the parameters. Even then, because some of the apparatus andphotosynthetic organisms drift over time, the comparison is far fromperfect. By contrast, the present invention mimics mass production in amore manageable way which allows for easier evaluation of parametercombinations.

The present invention may isolate key parameters and mimic theconditions in a mass production system to allow optimization fordifferent cultures of microorganisms. In mixotrophic culture the impactof light is less important than the contribution of growth energy andcarbon from the organic carbon source. The units in the describedinvention are well mixed which allows for a volumetric reaction rate.For the mixotrophic reactions, growth is dominated by volumetricreactions rather than areal reactions resultant from solar input.Artificial light or solar light may be supplemental, and the majority ofgrowth may be resultant from the organic carbon source.

Previous attempts to optimize parameters of a microorganism culture haverelied upon rather simple lab scale selections which do not resemblereal life situations, and thus the data produced does not translate toimproved cultures in commercial production conditions. For example,traditionally, optimal microorganism strains are typically selected bygrowing different strains in multiwelled plates or separate small volumevessels and measuring the results of either cell growth or productproduction. These multiwelled plates are not well mixed and do not allowfor a feedback mechanism with the consumption of carbon during thereaction given their small culture volume.

The control mechanisms for such a screening are different from thoseused in large scale production (i.e., commercial production). Further,this may not reflect actual results in mass production in a largebioreactor or even an open pond where parameters such as dissolvedgases, depth of the culture, and lighting conditions vary dramaticallyfrom the screening system. The traditional selection using small volumeswith poor mixing may not choose a desired optimal microorganism strainor culture parameters and may be more likely to select a strainoptimized for a batch system resembling the selection system rather thanany large scale continuous system.

During the operation of the array of vessels in the present invention,numerous sensors may be present with continuous monitoring of cultureparameters. This allows for a thorough determination of preferredparameters. For example, one set of parameters may be ideal for thefirst few days which destroy the culture on, for example, day seven.Without constant monitoring, one would assume this set of parameters isunacceptable when looking only at the final result, however the set ofparameter may be ideal provided that one make an adjustment on, forexample, day five or harvest the culture before day seven.

Another embodiment of the present invention is the determination of theapproximate cost and benefit of mass production using a photosyntheticmicroorganism in the bioreactor design stage before even building such aplant. Unlike conventional laboratory systems, the present inventionmimics actual large scale production conditions, permittingextrapolation and translation of useful data.

While considerable work has been conducted on phototrophic microalgaeincluding the optimization of parameters for growth, no systems arecommercially available to rapidly test mixotrophic cultures ofmicroalgae in parallel for rapid combinatorial screening of strains,process conditions, media, growth factors, organic carbon source,contamination vectors, and more. The number of parameters affectinggrowth and photosynthetic microorganism longevity are greater inmixotrophic systems than for phototrophic systems with the inclusion ofthe complexity of non-light energy sources, such as an organic carbonsource, and the ensuing challenges of contamination (e.g., bacteria,fungi, ciliates) that may feed on the organic carbon source.

The apparatus used in the present invention may be fitted with a wideselection of sensors. Preferred sensors may comprise: pH, temperature,dissolved oxygen (DO), dissolved carbon dioxide, cell density,concentrations of various chemicals in the medium, amount of light, andwavelengths of light received. A sample of culture medium may bewithdrawn for testing or the testing may be done on or inside theculture vessel itself. One example of a continuous flowing sensor ofnumerous components in water is exemplified by U.S. Pat. No. 8,102,518.

The apparatus may be operated in an axenic mode or may be operated in anon-axenic mode. An advantage to a non-axenic mode is the understandingof the impact of growth conditions on bacteria both in terms ofproliferation and individual strains of bacteria. The apparatus may beused to test the response in microalgae and contaminants to treatmentsapplied for increasing microalgae growth, longevity, or reducecontaminants.

While every individual vessel may have its own set of sensors, it may bebeneficial to have a single common sensor for each type of parameter.For example, by having a line to collect exhaust gases from each vesselseparately, the same sensor can provide repeated sequential measurementsfrom each gas stream. Likewise, for withdrawing a small sample of liquidand running it by a single sensor for each type of parameter beforereturning the sample to culture vessel. This avoids any difficulty ordrift in separate sensors.

The light may be provided to the cultures by any conventional lightingsource such as fluorescent bulbs, light emitting diodes (LEDs), ambientroom light, sunlight, etc. In some embodiments, the light source may bea lighting device exterior to the vessel. Reflected, refracted andfiltered light may also be used. In some embodiments, light may beprovided to the culture from inside the culture vessels by a submergedlight source such as fiber optics, LEDs or an LED strip or otherlighting device disposed within the interior volume of the culturevessel. Controlling lighting parameters such as the intensity, amount,duration, pulsing, light/dark cycle, wavelengths provided, etc., areparticularly desirable and these parameters may desirably change duringthe culturing process and in response to other changing parameters.

In one embodiment, light may be only applied to a portion of the vesselto evaluate the impact of reduced light, no light, high light, orpartial light on the growth and productivity of the targetmicroorganisms as well as unwanted species (i.e., contamination).

These and other parameters that are harmful to the photosyntheticmicroorganism may be used if desired to enhance product production orthe desirability of it. For example, at the end of the culture cycle,one may wish to dramatically increase the amount of light to photobleach(i.e., stress) the microorganism to aid in recovery of a product byautolysis or separation of the product.

The types of products that can be produced include, whole cells,extracts, lipids, proteins, pigments, hormones, polysaccharides, andothers. The amounts and proportions of each are frequently altered byaltering the process parameters. The product may be an action also suchas degradation of wastes or catalytic biotransformation of one chemicalto another.

Changing parameters during cultivation to have a desirable effect on thephotosynthetic microorganism's metabolism may be preferred. Whilelaboratory experimental examples are known of adding a chemical toenhance or stop production or growth, they are not typically done underconditions suitable for mass production due to a lack of understandingof the interactions and effects. The present invention would allow for asystematic evaluation of interactions through combinatorial testing andreduce risk for introducing chemicals or treatments at a masscultivation scale.

Supplies of gases to the culture vessels of the present invention may beprovided by a common line from a common reservoir. Individual valves andmeters may adjust one or more gasses before being optionally mixedtogether and supplied to the cultures. A bubbler or sparger at or nearthe bottom of the culture vessels may be used to deliver the gases andprovide mixing in the culture. Laminar flow of bubbles may be used toprovide mixing for shear sensitive microorganisms. If desired, mixingbaffles or a separate active mixer of any conventional type may beadded.

During the culturing, addition of any chemical and/or withdraw (andoptional recycle) of culture medium (with photosynthetic microorganisms)may be performed as a parameter. The culture may be run in batch, fedbatch, semi continuous or fully continuous methods.

A further embodiment is the use of a fed-batch mode of operation wherebythe organic carbon source may be slowly fed as the organisms grow. Theorganic carbon source may be used to control pH or may be addedsparingly to disfavor the competitive production of by-products, whichmay include bacteria or other unwanted microalgae species. The organiccarbon source may be concentrated or dilute. The volume of the cultureincreases with the addition of the organic carbon source. In someembodiments, the culture may be periodically harvested or continuouslyharvested with an overflow port that controls the volume to a presetlevel. The use of the parallel culturing vessels may be used toconcurrently test the different modes of harvest as a parameter thataffects growth and longevity.

While exemplified by a clear vessel, the vessel may comprise translucentor even opaque regions in part or in its entirety, with at least onesource for delivering light or feature allowing light to be delivered.For example, it may be desirable to have steel supporting structures incontact with the vessel or to have the vessel in contact with a metalheat sink for heating or cooling the vessel (i.e., heat exchange),either of which may block light from reaching the culture volume.

While the cultures are discussed as producing a product, the culture mayalso be used to degrade or remove undesirable substances from theculture medium. This would be especially desirable for optimizing theculture parameters to degrade wastewater or thrive on waste mediums. Theculture may also react with or catalytically transform one feedstockinto a more desired product during either growth or steady stateconditions in light or dark.

Some or all of the bioreactor vessels may be sealed which allows forpressurization and prevents outside contamination from entering thevessel.

After each batch or periodically if continuous, the bioreactor may beeasily disassembled for ease of cleaning. The array of vessels in thebioreactor may be individually removable for cleaning, analysis, productrecovery, or medium addition.

The present invention may also incorporate the use of disposable orrecyclable components that may comprise the use of a plastic bag toserve as the reaction vessel. A bag reaction vessel may be disposedafter conducting a desired experiment. The bag may be supported by astructure for frame to maintain the distance from the culture volume tothe light source.

For the purposes of this specification the term “clear” refers totransparent or translucent to light, particularly allowing thetransmission of the light wavelengths utilized for photosynthesis by thephotosynthetic microorganism. It is understood that even “clear” vesselswill have some light transmission loss that may range from about 1 to40%. The desirable wavelengths, such as photosynthetically activeradiation (PAR) light or light promoting production of a product, mayvary somewhat between different species of photosynthetic microalgae. A“clear” material may be opaque or hinder other wavelengths of visiblelight and other wavelengths of electromagnetic radiation. A “clear”material is clear only in the sense of its optical properties and onlyto an adequate degree for allowing light to pass through to thephotosynthetic microorganism.

In another embodiment of the present invention, the light source mayemit only certain wavelengths of light that have value in promotinggrowth or production of desired product. LEDs of a specific wavelengthor wavelength enhanced LEDs emitting light in the desired wavelengthsmay be used. The light source need not exclusively emit desiredwavelengths (e.g., PAR light) but rather may be enriched for desiredwavelengths in this embodiment of the invention. A number of differentelectro luminescence devices known per se may be used.

Also, an optical filter may be added between the light source and thephotosynthetic microorganism to block harmful wavelengths or wavelengthswhich reduce production of the desired product(s). Harmful wavelengthsmay even degrade the desired product(s) or degrade an intermediate inthe metabolic pathway of the desired product(s). Certain harmfulwavelengths may not be harmful to the photosynthetic microorganism butrather encourage a different metabolism away from maximum production ofthe desired product(s). The optical filter may be a device, thin film,particle, or simple compound, which reflects or adsorbs some of theundesired wavelengths or even neutral wavelengths. This filter may beoutside or inside a culture vessel.

Nutrients required by the photosynthetic microorganism, e.g. sodiumnitrate and sodium phosphate or others, may be added manually either inthe solid form by premeasured manual addition or may be added manuallyor automatically as a premeasured diluted solution in water via the topof the vessel or elsewhere in a line. These nutrients may also be addedwith the organic carbon media, which may be added continuously orsemi-continuously in response to changing parameters, such as pH orvolume and the like. The nutrients may be pure ingredients, knownmixtures or relatively raw materials such as the wastewater from a foodprocessing facility. All added products to the system are preferablysterile to limit the amount of contamination introduced into the culturevessel. The addition of CO₂ creates carbonic acid in water that willlower the pH during the cycle time of a batch, therefore the pH may beconstantly monitored (by testing a sample or by a pH sensor located inthe vessel or a line of circulating liquid) and a buffer or an alkalinematerial, such as sodium hydroxide or sodium bicarbonate etc., may beadded via the same technique as the nutrients to control the pH at thedesired level for optimum organism growth and performance. When thebioreactor is open vented, there may be no pressure other than theliquid head pressure in the vessel and the vessel may be run at or closeto the full volume level. The top of the vessel may be used for addingnutrients, mounting pH instrumentation, adding control chemicals, andperiodic internal cleanout of the reaction vessel and lines. Likewisefor a closed bioreactor, the top may also be used for the same featuresprovided that they are sealed with the vessel top.

A feature of the present invention is to have common reservoirs ofmaterials to add to the multiple culture vessels. They may use a commonline with manifolds and individual or group valves or adjustable metersto deliver the same or differing amounts to different vessels. Thisprovides further control of the amounts and concentrations added todifferent test vessels thereby providing better comparisons when one ormore parameters are changed.

Another feature may be to have automated control of the variousparameters based on preset programs or feedback loops from the sensors.Since many slightly different cultures of photosynthetic microorganismsmay be present, it may be impractical to monitor all of them separately.

Additionally, data may be collected for storage and comparison analysis.Having as many of the parameters controlled along with common datacollection makes for a better comparison. In the present invention, asimple run of the bioreactor apparatus may provide a considerable amountof comparable data for data mining by conventional statistical analysissuch as the statistical programs from SAS Institute Inc. (100 SAS CampusDrive, Cary, N.C. 27513-2414).

The concentrations of the nutrient and pH control solutions arecarefully selected to minimize damaging or killing the microorganisms,and to provide long term control during the growth and productmaturation phases (e.g., oil accumulation, pigment accumulation) of theprocess. Redundant pH probes may be included to provide easy switchoverto a new probe when the recording probe fails to operate. It is notedthat a system that filters the microorganisms and provides clean waterto the pH probes could extend the life of the probes in this service.

Contamination by undesired microorganisms may be reduced by adding aninhibitory gas, such as ozone, chlorine dioxide, ethylene oxide etc., inthe headspace or by the CO₂ sparger or a separate gas sparger. If aspecific contaminant is of particular concern, an antibiotic, which maybe less harmful to the desired photosynthetic organism, may be added tothe culture medium. Likewise, the culture conditions may be modified toinhibit the contaminant without excessive harm to the photosyntheticmicroorganism.

A preferred embodiment of the present invention is a bioreactor designfor changing conditions while culturing photosynthetic microorganisms.The bioreactor allows for a batch of microalgae or other microorganismto undergo several days of both continuous growth and an oil (or othermetabolite) buildup phase of production in batch or semi-batchconditions. Alternatively, continuous production may be used. Theculture conditions may alternate between enhancing growth of thephotosynthetic microorganism and enhancing production of a metabolite,such as a fatty acid or other desired compound(s). The culture may alsobe designed to degrade unwanted or harmful compounds in the medium.

The area outside the culture vessel may use an air ventilation system toremove the heat produced by the light bulbs and other equipment.

Computer controlled adjustable motors and valves may be responsive tothe culturing process conditions. Preset parameters and changes inparameters may be effected by computer control, by feedback loop, ormanually by an operator. Redundant manual or override controls may bepresent. Specific operations may comprise: the addition of pH controlagents, gases, liquids, nutrients; removal of culture liquid or gases;adjusting the agitation, recirculation, and gas introduction rates; andtemperature control. The operation may be run continuously andindefinitely to study the long-term effects or to test for and optimizemicroorganism longevity.

At the end of a cycle, or continuously, when microorganisms are removedfrom the system, the desired product(s) may be extracted from themicroorganism in a separate downstream process such as, but not limitedto, solvent extraction, supercritical fluid extraction, and celldisruption. The method of extraction and recovery will depend on theparticular product(s) targeted, and are preferably done by a mannerknown per se.

Example 1: The Bioreactor Array

A set of identical 1 liter glass culture vessels having a workingculture volume of 500-800 ml and columns with dimensions of 4.57 cminside diameter (ID)×5.1 cm outside diameter (OD)×61.0 cm height (H)(1.8″ ID×2.0″ OD×24″ H). A rubber stopper is pressed into the open topof the column to serve as a lid. Holes were cut into the rubber stopperto accommodate a 0.762 OD×66.04 cm (0.3″ OD×26″H) glass capillary tubefor aeration, a 0.601 cm OD×30.48 cm H (0.24″ OD×12″H) pH probe (otherdesigns employed a shorter pH probe (12 mm×152 mm)), a sample/fill portvia 0.476 cm OD×0.159 cm ID Tygon tubing ( 3/16″ OD× 1/16″ ID), anorganic carbon liquid (e.g., acetic acid) injection via 0.3 cm OD×0.1 cmID Tygon tubing (0.117″ OD×0.039″ ID), a thermistor 0.0.635 cm OD×3.81cm L @30.48 cm depth (0.25″ OD×1.5″L @12″ depth), and a 0.254 cm (0.1″)hole for venting. The probes were placed at a position to be submergedinto the culture medium contained in the glass culture vessels. A 24V DCperistaltic pump rated at 1 ml/min flow was used to pump organic carbonliquid to the columns. A 0-10Ω potentiometer was used to control theperistaltic pump speed. The pump was actuated using a signal from aHanna pH controller (these controllers have a hysteresis of 0.1). A 50ml polypropylene centrifuge tube was used as the acetic acid reservoir.Aeration was controlled via a rotameter (0.7 L/min maximum flow). A LuerLock was placed on the end of the sample/fill Tygon tubing. Lighting wasprovided via an 8 bulb T5 fluorescent bulb light fixture. Thefluorescent bulbs apply light from about 50 to 500 microeinsteins/m²s.The axes of the light tubes were aligned perpendicular to the verticalaxis of the each culture vessel for this specific unit. Alternatively,the lights can be aligned parallel to the vertical axis of each culturevessel. A box fan was placed adjacent to the lights blowing toward thecolumns to help remove some of the heat from the lights. Data logging isaccomplished by way of a C-RIO and NI software

Initially there were no controls for the CO₂; it was fed at a slowconstant rate (app. 0.02 LPM). An optional water bath type coolingsystem made of a 2 ft by 2 ft (61 cm by 61 cm) flat panel made of clearacrylic was used as the bath. A submersible pump was placed into aninsulated reservoir and pumped water through a 2400 btu/h chiller on itsway to the water bath. From the water bath water flowed back to thereservoir. The water bath system is able to hold temperatures to 20° C.(no heating capability).

Additional Bioreactor Array Embodiments

An alternative system may mount each culture vessel on an aluminum blockwith a Peltier temperature control system, which is independentlyvariable instead of a water bath.

Another bioreactor array design may comprise multiple variations. LEDlights may be added in place of the fluorescent lights and use multiplediodes (e.g., red, far red, blue) with wavelengths that range from about300 to about 800 nm. Each diode may be dimmed and variably controlledindependent of one another. A second modification may be a set ofoverflow ports placed at 600, 700, and 800 ml on each 1000 ml column. Anoverflow reservoir may be added and plumbed to the desired overflowports. The unused ports may be capped.

In another bioreactor array embodiment, the water bath cooling systemmay be replaced by thermoelectric cooling. The column stand for thissystem may be fully enclosed with two clear plexiglass faces in frontand back for viewing and lighting the columns. The top, bottom, andsides may be constructed from 0.75 inch (2 cm) plywood to help insulatethe chamber. The chamber may be split down the center with 0.75 inch (2cm) plywood creating two chambers that house 4 column reactors and 4acid reservoirs per chamber. Thermoelectric coolers (app. 80-100 btu/h)may be added to each chamber and controlled via simple thermostat. LEDlights may be employed in place of the fluorescent lights. These lightsmay have multiple diodes (e.g., red, blue, and white diodes) that mayindependently controlled as for intensity, photoperiod, and otherparameters. A variable voltage power supply may be used to power theliquid delivery pumps. This allows the pumps to be sped up or sloweddown easily with no need for resistors. Rotameters may be added andplumbed into the sparger air to facilitate the use of an additional gas.

Example 2: Control of the Bioreactor Array

The bioreactor array system of Example 1 was fitted with a programmablelogic controller (PLC) controls/data logging system. The PLC is used tocontrol temperature, pH, dissolved oxygen (DO), lightintensity/spectrum, light cycle, and growth mode (phototrophic,heterotrophic, mixotrophic) all of which can be manipulated through auser friendly touch screen mounted directly to the unit. This systemuses Peltier devices for heating/cooling, which can be used for climatecontrol or can be mounted to the column directly for individualtemperature control. Controls for CO₂ as well as acid or alkaliinjection for pH control are used. There are multiple versions ofharvest systems that can be employed on this system (e.g., manual,overflow, pumped) depending on operator choice. An inert gas can beadded to sparger gas to help control DO via a feed back control loopbetween a DO probe and solenoid to control gas flow. A continuous mediaaddition system can be added for concentration control and are driven bya signal from an optical density type sensor. Media and acid consumptionare monitored by load sensors installed on the reservoirs.

Example 3: Bioreactor Array Operation

The column bioreactor array system of Example 1 is used to determinewhether an antimicrobial gas treatment can prevent or treat culturesthat were contaminated. A single Chlorella sp. strain is added to allvessels along with conventional BG-11 growth medium at a concentrationof 1 g/l. A harmful contaminant Polyarthra vulgaris, is added to eachculture vessel at the designated time shown in Table 1. Variousconcentrations of antimicrobial gases (i.e., ozone, chlorine dioxide,ethylene oxide) are mixed with carbon dioxide enriched air and bubbledthrough the culture vessels. The array of vessels are grown for a 10 daycycle under 12 hours light/12 hours darkness for 5 days usingnutrient-sufficient medium (growth phase) followed by 5 days without anitrogen source in the medium (oil accumulation phase). The conditionsand all other parameters are held constant with a common light sourceand common lines delivering the same amounts of solids, liquids andgases to each. The combinations of culture treatments are given in Table1.

TABLE 1 Culture vessel Time of adding number Gas treatment Gasconcentration contaminant 1 (control) None None Initial 2 (control) NoneNone 3 days 3 (control) None None 8 days  4 Ozone Low Initial  5 OzoneLow 3 days  6 Ozone Low 8 days  7 Ozone Intermediate Initial  8 OzoneIntermediate 3 days  9 Ozone Intermediate 8 days 10 Ozone High Initial11 Ozone High 3 days 12 Ozone High 8 days 13 Chlorine dioxide LowInitial 14 Chlorine dioxide Low 3 days 15 Chlorine dioxide Low 8 days 16Chlorine dioxide Intermediate Initial 17 Chlorine dioxide Intermediate 3days 18 Chlorine dioxide Intermediate 8 days 19 Chlorine dioxide HighInitial 20 Chlorine dioxide High 3 days 21 Chlorine dioxide High 8 days22 Ethylene oxide Low Initial 23 Ethylene oxide Low 3 days 24 Ethyleneoxide Low 8 days 25 Ethylene oxide Intermediate Initial 26 Ethyleneoxide Intermediate 3 days 27 Ethylene oxide Intermediate 8 days 28Ethylene oxide High Initial 29 Ethylene oxide High 3 days 30 Ethyleneoxide High 8 days 31 Ozone Low None 32 Ozone Intermediate None 33 OzoneHigh None 34 Chlorine dioxide Low None 35 Chlorine dioxide IntermediateNone 36 Chlorine dioxide High None 37 Ethylene oxide Low None 38Ethylene oxide Intermediate None 39 Ethylene oxide High None 40(control) None None None

The differing gas treatments without adding a contaminant serve as acontrol for determining the baseline of inhibitory effects of the gas onthe microalgae. Likewise the differing time for inoculation with thecontaminant serve to determine a baseline of the harmful effects on theculture depending on the growth cycle. The differing gasses and theirdiffering concentrations serve as techniques being optimized between theharmful effects on the microalgae and the beneficial harmful effects onthe contaminating microbe.

At the end of the 10 day cycle the resulting culture liquid iscentrifuged and dried, the biomass weighed, the protein contentestimated by the Comassie Blue method and the lipid content estimated bythe Nile Red method (Cooksey et al, (1987)). The entire process can becompleted in two weeks as compared to a year or more using a singlesystem with the potential for changing conditions (especially lightconditions) and instrument drift during that time.

The method may be repeated with any combination of photosyntheticmicroorganisms and contaminant and may be repeated where theantimicrobial gas is not added until after the contaminant has beenadded and is detectably affecting the cell growth. This approach maydetermine whether any antimicrobial gas treatment can rescue acontaminated culture. Further methods may be run with other combinationsof microorganisms and varied parameters to draw other conclusions in asimilar manner and short time frame.

Previous systems using the 2 ft (61 cm) by 2 ft (61 cm) flat panelreactors with a volume of 10-15 L required about 15 g of biomass toinoculate each reactor. Combinatorial experiments using eight reactorswould require about 120 g of biomass and about 120 L of prepared media.In one embodiment of bioreactor array system, the culture vessel mayhave an operating volume of 500 to 1000 ml, which requires only 0.5-1.5g, or about 1 g, of biomass to inoculate each reactor. Combinatorialexperiments using eight culture vessels of the instant invention wouldrequire about 8 g of biomass and about 4-8 L of prepared media. Thedramatic reduction in biomass and culture media resources requiredallows for more experiments to be done, but still produces the minimumamount of biomass required for composition analytical tests to beperformed. The results from the composition analytical tests may be usedfor product development and verification of parameters for larger scalebioreactors.

The capability for each culturing vessel in the bioreactor array to behave individual and independently controllable organic carbon, lighting,and gas supply systems provides the flexibility to operate inphototrophic, mixotrophic, and heterotrophic culturing conditions. Insome embodiments, the culturing vessel may receive light and air orcarbon dioxide gas, but no organic carbon to operate in phototrophicculture conditions. In some embodiments, the culturing vessel mayreceive light, organic carbon, and gases (e.g., air, carbon dioxide,oxygen) to operate in mixotrophic culture conditions. In someembodiments, the culturing vessel may receive organic carbon and oxygenor air, but no light to operate in heterotrophic conditions. Parametersmay be independently controlled in the bioreactor array as describedthrough the specification to produce biomass and combinatorial testingresults in any of the described culture conditions. Dissolved oxygen(DO) control becomes important in mixotrophic and heterotrophicconditions due to the consumption of oxygen by the microorganisms andhigh cell densities that may result from rapid growth. An inert gas maybe added to the sparger air to help control the dissolved oxygen via aDO probe and a solenoid to control gas flow.

Multiple available harvest systems may comprise a manual harvest system,an overflow harvested system set a desired volume (e.g., 600 ml, 700 ml,800 ml), or a pumped harvest system. The harvest systems may becontrolled for different harvesting rates from the different culturevessels by methods such as, but not limited to: controlling theharvesting pump devices at different settings, and setting the overflowvolume level at different volume levels in different culture vessels.Continuous media addition may be added for cell concentration controlindependently of the harvest system or in combination with the harvestsystem, and may be driven by a signal from an optical density sensor.

In one embodiment the bioreactor array may comprise a plurality ofculture vessels configured to contain an aqueous culture ofmicroorganisms in an interior volume of the culture vessel. Each culturevessel in the bioreactor array may comprise an independently controlledsupply of gas, nutrients, and light. The gases may comprise air, oxygen,carbon dioxide, nitrogen, and/or other inert gases. The supply of gasesmay be controlled to maintain levels of dissolved carbon dioxide,dissolved oxygen, pH, and mixing by aeration. The supply may becontrolled as to the type of gas, volume of gas, concentration of gas,flow rate, and bubble size. The nutrients may comprise minerals in anaqueous medium and/or an organic carbon source. The light may besupplied by lighting devices disposed outside the culture vessel, withinthe interior volume of the culture vessel, or combinations thereof. Theindependently controlled light supply may be completely turned off forheterotrophic culture conditions, or varied in amount, intensity,photoperiod, light/dark cycle, and wavelength of light for phototrophicand mixotrophic culture conditions. In some embodiments, the lightingdevice may comprise at least two light emitting diodes (LEDs) thatprovide different wavelengths of light and are independentlycontrollable.

In some embodiments, each culture vessel may further comprise anindependently controllable heat exchanger to control temperature of theculture. In some embodiments, the culture vessel may be translucent,transparent or clear, which facilitates the use of a lighting devicedisposed outside of the culture vessel. In some embodiments, the culturevessel comprises at least one opaque section which blocks light exteriorto the culture vessel. A culture vessel with at least one opaque sectionmay reduce light for mixotrophic conditions, block all light forheterotrophic conditions, or facilitate the use of a lighting devicedisposed within the interior volume of the culture vessel.

In some embodiments, the bioreactor array may further comprise anindependently controlled harvesting system for each culture vessel.Multiple available harvest systems may comprise a manual harvest system,an overflow harvested system set a desired volume (e.g., 600 ml, 700 ml,800 ml), or a pumped harvest system. The harvest systems may becontrolled for different harvesting rates from the different culturevessels by methods such as, but not limited to: controlling theharvesting pump devices at different settings, and setting the overflowvolume level at different volume levels in different culture vessels.Continuous media addition may be added for concentration controlindependently of the harvest system or in combination with the harvestsystem, and may be driven by a signal from an optical density sensor.

In some embodiments, the bioreactor array further comprises at least onesensor configured to measure at least one culturing parameter of eachculture vessel. The at least one sensor may be selected from the groupconsisting of: pH sensor, temperature sensors, light sensors, dissolvedoxygen sensors, dissolved carbon dioxide sensor, and optical densitysensor. Sensors at each culturing vessel may be coordinated for controlof the other features of the bioreactor array through a programmablelogic controller, and for data recording through a data logging device.

Embodiments of the bioreactor array may be used in methods of culturingmicroorganisms, particularly for combinatorial testing of differentparameters for cultures of the same microorganisms, the same parametersfor cultures of different microorganisms, and combinations thereof. Inone embodiment, a method comprises providing a culture of microorganismsin an aqueous culture medium in a plurality of culture vessels;independently controlling the supply of at least one selected from thegroup consisting of light, at least one gas, at least one nutrient, andheat exchange to each culture vessel; and wherein each culture vesselcontains a culture of the same microorganisms with different parameters.The parameters may be selected from the group consisting of temperature,pH, amount of light, intensity of light, wavelengths of light, lightphotoperiod, light/dark cycle, concentration of gases, and agitationfrom gas supply. For embodiments with harvesting systems, the harvestingrate may also be an independently controlled parameter for each culturevessel.

It will be understood that various modifications may be made to theembodiments disclosed herein. Therefore, the above description shouldnot be construed as limiting, but merely as exemplifications ofpreferred embodiments. Those skilled in the art will envision othermodifications within the scope and spirit of the claims appended hereto.All patents and references cited herein are explicitly incorporated byreference in their entirety.

1-20. (canceled)
 21. A method for evaluating the feasibility of largescale microalgae culture, comprising: a. providing a plurality ofmicroalgae cultures in an array of closed culturing vessels, wherein (1)each vessel of the array comprises (a) a plurality of sensors selectedfrom the group consisting of a pH sensor, a temperature sensor, a lightsensor, a dissolved oxygen sensor, a dissolved carbon dioxide sensor,and an optical density sensor and (b) a plurality of controls selectedfrom controls for pH, light, gas levels, media levels, temperature,culture agitation, and culture harvesting; (b) the culturing vessels arelinked to one or more shared reservoirs for controllably adding culturemedia, gas, or both to the vessels; and (c) each culture comprises atleast 0.5 grams of microalgae in a volume of at least 500 ml of anaqueous culture medium; b. independently subjecting each microalgaeculture to one or more related test conditions wherein at least aplurality of the cultures is subjected to different degrees of at leastone test condition; and c. evaluating the impact of the test conditionson the cultures to determine the effect of the test conditions on themicroalgae and assessing the feasibility of performing large scalecommercial microalgae culture under one or more production conditionsrelated to the test conditions from the results of the evaluation. 22.The method of claim 21, wherein the method comprises contemporaneouslyculturing a plurality of test cultures and a plurality of controlcultures in the array.
 23. The method of claim 22, wherein the methodcomprises contemporaneously culturing a plurality of cultures under thesame conditions such that this plurality of cultures serve asrepetitions in an experiment.
 23. The method of claim 22, wherein themethod comprises contemporaneously culturing at least three controlcultures and at least three test cultures.
 24. The method of claim 23,wherein the method comprises contemporaneously culturing a plurality ofcultures under the same conditions such that this plurality of culturesserve as repetitions in an experiment.
 25. The method of claim 23,wherein the method comprises contemporaneously testing three or moredifferent conditions in the array by culturing three or more cultureswherein each culture of the three or more cultures is cultured under adifferent condition.
 26. The method of claim 24, wherein the methodcomprises culturing multiple cultures for each of the three or moredifferent conditions being tested in the array, wherein each culturebeing tested for a condition is cultured under different levels of thecondition.
 27. The method of claim 26, wherein the different conditionscomprise culturing the microalgae in the presence of differentchemicals.
 28. The method of claim 26, wherein the method comprisescontemporaneously testing five or more different conditions in thearray.
 29. The method of claim 21, wherein the method comprises addinggas, media, or both, to a plurality of the cultures during the cultureprocess from a shared media reservoir, gas reservoir, or both.
 30. Themethod of claim 29, wherein the method comprises supplying gas,supplying media, or both, to one or more vessels by passing the gas,media, or both through a lid that encloses the top of the vessel. 31.The method of claim 25, wherein the method comprises adding gas, media,or both, to a plurality of the cultures during the culture process froma shared media reservoir, gas reservoir, or both.
 32. The method ofclaim 31, wherein the method comprises supplying gas, supplying media,or both, to one or more vessels by passing the gas, media, or boththrough a lid that encloses the top of the vessel.
 33. A system fortesting the feasibility of large scale culture of microalgae underdifferent conditions comprising: (a) an array of at least eightsubstantially identical culturing vessels which are suitable forculturing microalgae, wherein each vessel is capable of containing aculture of at least 500 mL in volume and comprises (i) a plurality ofsensors selected from the group consisting of a pH sensor, a temperaturesensor, a light sensor, a dissolved oxygen sensor, a dissolved carbondioxide sensor, and an optical density sensor and (ii) a plurality ofcontrols selected from controls for pH, light, gas levels, media levels,temperature, culture agitation, and culture harvesting; (b) a shared gasreservoir and delivery system, which is connected to each of theculturing vessels in the array and is capable of independently andcontrollably adding gas to each of the vessels; (c) a shared mediareservoir and delivery system, which is connected to each of theculturing vessels in the array and is capable of independently andcontrollably adding media to each of the vessels; and (d) a programmablelogic controller that controls at least a plurality of the controls andreceives data from at least a plurality of the sensors in each of thecultures.
 34. The system of claim 33, wherein more than one of theculture vessels is light transmissive.
 35. The system of claim 34,wherein each of the culture vessels comprises a lid through which a gassupply inlet and a nutrient supply inlet passes.